This proposal aims to develop a collaboration between IDX Labs and the University of Virginia that will take real-time PCR tests for Cryptosporidium and Giardia from the research arena to the clinical lab. These intestinal protozoa cause diarrhea worldwide, with an estimated 2 million infections per year in the United States. The investigators in this proposal have developed and patented real time PCR tests for (1) Giardia that sub-genotypes into the major Assemblages A and B and (2) Cryptosporidium that speciate the parasite into the major pathogens Cryptosporidium hominis, parvum, and meleagridis. The tests are robust, with comparable sensitivity vs. gold-standard antigen detection by ELISA, and have been validated on over 400 patients from Bangladesh and Tanzania. However the tests have been designed for research purposes and require modifications to make them widely usable in clinical care. In aim 1 of this proposal we will optimize our DNA extraction method from stool into a high-throughput magnetic bead format involving Giardia and Cryptosporidium-specific DNA capture at downstream portions of the amplified 18S rRNA gene; this method provides both increased specificity (by requiring specific sequence for binding) and sensitivity (via elution of DNA away from PCR inhibitory substances in stool). In Aim 2 we will convert our individual genotype- specific PCR assays into a two-color single-vessel reaction that detects and distinguishes Cryptosporidium and Giardia without genotype information, which in the short-term remains clinically irrelevant for the low endemicity U.S. scenario. The reaction will continue to utilize Scorpion probes, a powerful technology for detection of DNA in stool because of the dual specificity provided at both primer and probe. We will adapt this new single-vessel test onto a commercial real-time PCR platform (e.g., Cepheid's SmartCycler). The innovation of this proposal lies not in PCR, but in the widely translatable DNA purification technology and in the concept of bringing the emerging field of molecular diagnostics to underfunded small-market infectious diseases. A Phase II application is anticipated to validate the final diagnostic test product against immunoassay for an FDA 501 (k) medical device application. [unreadable] [unreadable] [unreadable]